Each year, billions of gallons of metalworking fluids (MWFs) are consumed in manufacturing and a number of human health effects have been associated with exposure to MWFs, including various respiratory diseases, skin conditions, and cancers. Microbial contamination of MWFs has emerged as an important hazard to exposed workers. A newly identified organism, Mycobacterium immunogenum, appears to have an etiologic role in hypersensitivity pneumonitis (HP) in relation to metalworking fluids. However, our knowledge of this organism and the microbial ecology of MWFs in general is limited. Our preliminary studies reveal the wide-spread and high abundant presence of Mycobacteria in MWFs-associated biofilm samples from an automobile plant while no Mycobacteria were detected in some of the corresponding bulk fluid samples, which strongly support our hypothesis that biofilms are favorable sites for Mycobacteria to grow and survive in MWFs. In this exploratory/developmental project, we will prospectively characterize the microbial ecology of MWFs, with a specific focus on biofilms, Mycobacteria and Pseudomonads in metalworking fluids, and identify factors critical to the growth and survival of Mycobacteria in MWFs. Our specific aims are (1), Characterize microbial communities in MWFs and associated biofilms using molecular approaches;and (2), Monitor the prevalence and dynamics of Mycobacteria and Pseudomonads in MWFs using real-time PCR and correlate and identify parameters that contribute to the dynamics of Mycobacteria and Pseudomonads. . This proposed research will apply novel approaches (e.g., a major focus on biofilms associated with MWFs) and molecular techniques (e.g., 16S/18S rRNA clone library and real-time PCR) to better understand the overall microbial ecology of MWFs, and in particular, dynamics of Mycobacteria and Pseudomonads in MWFs. Statistical tools will be used to help to identify physical or chemical parameters that contribute to the growth of in Mycobacteria and/or Pseudomonads MWFs. The data obtained from this developmental project will enable future projects to develop novel approaches for the control of biofilm growth and Mycobacteria and Pseudomonads in MWFs. The outcome from this study and future projects will help users of MWFs to identify the right targets and develop novel approaches to monitor, control, reduce and/or prevent exposures to MWF-associated microbes that are suspected to cause adverse human health effects. Ultimately, this would serve to reduce the risk of adverse health events related to inhalation exposure to microbes or microbial products in MWFs. Public Health Relevance: We are combining both molecular methods and statistical tools for a prospective study of microbial ecology of metalworking fluids with a special focus on biofilms, Mycobacteria and Pseudomonads. The project responds up front to an immediate challenge to occupational health, i.e. hyposensitivity pneumonitis among workers exposed to Mycobacteria and Pseudomonads growing in metalworking fluids. Success of this project will have a great impact on occupational and public health.